Nucleus Accumbens D2-MSN activity is thought to drive reward or block aversion. However, recent work indicates D2-MSNs play a more complicated role in encoding information and can drive reward to natural stimuli. Additionally, D1-MSN and D2-MSN activity is differential and often opposing due to dopamine receptor expression and lateral inhibition mechanisms. Our work shows D1-MSNs enhance excitation on D2-MSNs through peptide release and ChI firing. This potentiation is sub-region specific and we predict this specificity is due to differential input to these regions. Using whole-cell slice physiology and combined optogenetics, we are first determining if specific inputs respond to substance P differentially by examining potentiation of multiple inputs (ventral hippocampus, paraventricular thalamic nucleus, and the agranular insula). We hypothesize that multiple inputs can integrate and promote peptide release. Therefore, we are probing excitatory inputs to MSNs in the NAc core using a dual optogenetic approach with red-shifted opsins to determine if strong stimulation of these inputs can evoke peptide release and excitatory potentiation on D2-MSNs. Additionally, we will be using a dual optogenetics and calcium imaging approach to examine single cell activity of both cholinergic interneurons and D2-MSNs using a miniscope implantation. Cholinergic activity is necessary for reward responding but it is unknown what long-lasting increases in ChI activity does to behavior. To activate ChIs, we will inject a bi-stable opsin that has long kinetics and mimics the response of substance P activation. We will use a conditioned food aversion task in which aversive shocks are paired with a novel palatable food distinct from animals home chow. We predict lasting ChI activation and D2-MSN potentiation will enhance associated learning of food and an aversive stimulus. Finally, using stimulation paradigms developed as discussed previously and the behavioral task described, we will examine how naturalistic in vivo activation of NAc ChI cells by excitatory inputs drive aversive behavioral responding.